基于SSR标记探讨三种金花茶植物的遗传多样性和遗传结构

薄叶金花茶、小花金花茶和小瓣金花茶是三种濒危金花茶植物,为了解珍稀濒危植物遗传多样性和遗传结构,该研究利用微卫星标记对他们的7个种群共184个个体进行了遗传多样性和遗传结构分析。结果表明:11个位点共检测到等位基因92个。在物种水平上,小瓣金花茶平均等位基因数(N_A)为3.9、有效等位基因数(N_E)为2.328、观测杂合度(H_o)为0.520、期望杂合度(H_e)为0.501,高于薄叶金花茶和小花金花茶。在种群水平上,有效等位基因数(N_E)在1.788～2.466之间,期望杂合度(H_e)在0.379～0.543之间;种群间遗传分化系数(FST)在0.143 7～0.453 3之间,种群间基因流(N_m)在0.301 5～1.488 9之间。AMOVA分子变异分析显示65.72%的变异存在于种群内。三种金花茶具有较低水平的遗传多样性和高水平的种群间遗传分化。STRUCTURE和PCoA种群遗传结构分析结果将取样种群分为2组,即薄叶金花茶和小花金花茶大部分个体分为一组,小瓣金花茶大部分个体分为一组。现存所有种群应根据实际情况尽快采取就地保护或迁地保护措施。     

濒危物种四合木与其近缘种霸王遗传多样性的比较研究

 采用垂直板聚丙烯酰胺凝胶电泳技术,对西鄂尔多斯高原的濒危植物四合木(Tetraena mongolica)种群和生长于同一生境的普通种霸王(Zygophyllon xanthoxylon)种群的遗传多样性和遗传分化进行了比较研究。等位酶分析表明,与其他灌木相比,两种植物均维持较高的遗传多样性。但濒危植物四合木种群遗传多样性水平低于霸王种群,多态位点百分率P分别为60％和83.3％,等位基因平均数A为1.6和2.2,平均期望杂合度He为0.245和0.392。种群间遗传分化不明显,基因分化系数Gst为0.051和0.020。这一结果说明不同类型的濒危植物具有不同的遗传含义,采取的保护措施也不尽相同。     

青钱柳种质资源多样性SRAP初步分析

为了利用分子标记方法评价青钱柳种质资源的遗传多样性,本文对青钱柳DNA的提取、SRAP扩增体系重要参数进行了优化,运用优化体系筛选多态性引物,并用1对引物对青钱柳9个种源进行了遗传多样性初步分析.研究结果建立了适于青钱柳SRAP的扩增体系;从110对SRAP引物中筛选出了13对多态性引物,运用1对引物组合Me7+Em2扩增获得21个多态性位点,多态率达100%.遗传多样性分析表明有效等位基因数(Ne)为1.342 9,平均Shannon's信息指数(I)为0.368 7,Nei's基因多样性指数(H)为0.226 7,种源的遗传分化指数Gst为0.198 3;聚类分析结果表明9个种源在遗传距离0.100处聚为3类,聚类结果和地理距离之间呈现较高的相关性.本文的研究结果表明所建立的青钱柳种质资源库具有广泛的遗传多样性,为进一步的开发利用提供了条件.     

濒危灌木长叶红砂遗传多样性的RAPD分析

应用RAPD分子标记对濒危灌木长叶红砂(Reaumuria trigyna)种群遗传多样性进行了分析.应用18条随机引物对长叶红砂5个种群的95个个体进行扩增,检测到118个位点,其中多态位点105个.结果表明:长叶红砂种群的多态位点比率(P)为88.98%,显示出长叶红砂种群存在较高的遗传多样性.Shannon多样性指数(0.4966)、Nei基因多样性指数(0.3303)和基因分化系数(Gst=0.1425)的分析结果显示,长叶红砂种群遗传变异大多存在于种群内,种群间的遗传分化占14.25%.聚类分析表明,长叶红砂种群遗传距离与地理距离之间无直接相关关系.遗传多样性水平与物种特性和所处不同群落有关,濒危植物并不一定表现为遗传变异水平的降低.     

水松自然种群和人工种群遗传多样性比较

采用ISSR分子标记技术分析水松不同起源种群的遗传多样性.结果表明:10条引物共检测出95个扩增位点,多态位点数占39.0%.与其他濒危裸子植物相比,水松的遗传多样性较低,遗传分化系数Gst为0.3982,基因流Nm仅0.3778,种群间存在一定程度的遗传分化,但种群内变异占主导地位;遗传距离与地理距离呈正相关关系.自然种群的多态位点百分率(P)、Nei的条带多样度(He)和Shannon信息指数(Ⅰ)平均值(39.3%、0.1499和0.2202)分别高于人工种群(30.7%、0.1265和0.1759).自然种群的遗传分化系数(Gst0.4513)和平均遗传距离(D=0.0301)也高于人工种群(Gst=0.3025,D=0.0192).     

岛屿特有种全缘冬青遗传多样性的ISSR分析

全缘冬青(Ilex integra)为岛屿特有植物,主要分布于浙江舟山群岛,被列为浙江省珍稀濒危植物。作者运用ISSR分子标记对舟山群岛的6个全缘冬青种群(5个自然种群和1个栽培种群)共57个个体进行了遗传多样性分析。采用9条随机引物扩增出78条清晰谱带,其中45条为多态性条带,多态位点百分比(PPL)为57.7%。经POPGENE软件分析,全缘冬青种群平均多态位点百分比(PPL)为42.1%,Nei’s基因多样度(HE)为0.153,较其他海岛植物的遗传多样性偏低。5个自然种群遗传分化系数Gst=0.316,AMOVA测度Fst=0.295,Shannon多样性分化系数S=0.304,遗传分化程度较高。全缘冬青种群间地理距离与遗传距离具有正相关性(r=0.649,P<0.05),岛屿间地理隔离对种群间遗传分化产生了较为显著的影响。UPGMA聚类分析显示,朱家尖岛与普陀岛种群亲缘关系较近,而舟山岛栽培种群是由桃花岛自然种群移植而来。针对全缘冬青种群遗传多样性较低及遗传分化程度较高的现状,应加强现有自然种群的就地保护,促进种群自然更新;建立种质资源库,收集不同岛屿的种源进行混合繁殖,促进基因交流。     

从松属相关物种筛选红松微卫星标记及其种群遗传多样性分析

近缘物种筛选法是获取微卫星引物的主要方法之一。利用松属植物EST-SSRs的种间通用性,从马尾松微卫星引物中筛选出红松EST-SSR标记,并与已有的红松基因组SSR标记进行对比分析。采集长白山露水河红松母树林4个种群的248个样本,分析种群遗传多样性。结果表明:马尾松与红松的种间通用率为27.27%,松属内不同亚属物种间的种间通用性偏低;与红松基因组SSRs相比,EST-SSRs的单位点等位基因数(N a)、有效等位基因数(Ne)、观测杂合度(Ho)、期望杂合度(He)等遗传多样性参数均较低;用2组引物分析4个种群的遗传多样性,发现有效等位基因均能反映出不同种群遗传变异的趋势,基因多样性均能反映出遗传同一性的差异,说明两组引物都能准确地反映红松种群的遗传多态性水平,新筛选的EST-SSRs可以用于红松种群遗传多样性研究。     

山茶科3种中国特有濒危植物的遗传多样性研究

猪血木(Euryodendron excelsum)、圆籽荷(Apterosperma oblata)和杜鹃红山茶(Camellia changii)均为我国特有的山茶科珍稀濒危植物。本研究应用随机扩增多态性DNA(RAPD)检测方法,比较了这3个物种的遗传多样性。利用20－24个随机引物从猪血木、圆籽荷和杜鹃红山茶居群中分别获得206－305条RAPD谱带,多态位点百分率(PPL)分别为51.80％、80.26％和38.83％;居群间遗传分化指数G_ST分别为0.3566、0.1713、0.1242。结果显示：自然分布较广的圆籽荷的遗传多样性高于另外两种自然分布区狭窄的种类。3个物种的遗传变异均主要存在于居群内,但猪血木居群间的遗传分化明显高于圆籽荷和杜鹃红山茶,这可能与其生境的严重片断化有关。根据3个物种不同的遗传结构特点,建议加强现有自然居群的就地 保护,促进居群自然更新;收集不同分布点的种源,尽快建立广东山茶科珍稀濒危植物的保育中心,深入开展人工快繁研究,扩大这些种群的数量。     

青鱼野生与养殖群体遗传变异的微卫星分析

研究利用自主开发的12个微卫星标记, 对来自于长江水系4个野生群体(湖北石首、湖南湘江、江苏邗江、浙江嘉兴)和1个养殖群体(江苏吴江)青鱼(Mylopharyngodon piceus)进行遗传多样性和遗传结构分析。遗传多样性分析结果显示这12个位点多态信息含量(PIC)介于0.660—0.923, 表明这12个位点均具有高度多态性(PIC>0.5)。5个群体的平均等位基因数(N_a)介于7.917—11.667, 平均有效等位基因数(N_e)介于4.837—6.035; 平均观测杂合度介于0.713—0.861; 平均期望杂合度介于0.749—0.819; 平均多态信息含量介于0.711—0.788, 表明这5个群体均具有较高的遗传多样性。遗传距离分析结果表明4个野生群体之间遗传距离较近, 养殖群体与这4个野生群体遗传距离均较远。UPGMA系统进化树显示, 湘江群体和石首群体首先聚为一支, 然后与邗江群体和嘉兴群体聚为一支, 最后与吴江养殖群体聚为一支。这12个微卫星位点具有较为丰富的遗传多样性特征, 可以用于青鱼不同群体的种质资源的评估和遗传多样性的分析。     

四川西部濒危植物桃儿七遗传多样性的RAPD分析

桃儿七是一种具有重要药用价值的珍稀濒危植物。采用RAPD分子标记技术,对在四川西部地区的桃儿七7个自然种群的遗传多样性水平和遗传结构进行了分析。用12个RAPD引物对7个种群共140个样品进行了扩增,共得到111条清晰带,其中32条具有多态性,在物种水平上多态位点百分率(PPB)为28.83%,在种群内的多态位点百分率变动幅度为4.50%至16.22%。同其它一些濒危植物相比,桃儿七种群具有较低的遗传多样性(He=0.0622,Ho=0.0987)。7个自然种群间出现了很强的遗传分化,分化指数接近70%。种群间的基因流低(Nm=0.2240)。造成上述结果的可能原因是与桃儿七的繁育方式和有限的基因流等因素有关。应将遗传多样性相对较高的松潘县牟尼沟种群作为原位保护的核心种群进行保护,尽量保护所有现有种群。     

Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP

Dendrobium officinale is a critically endangered perennial herb endemic to China. Determining the levels of genetic diversity and patterns of population genetic structure of this species would assist in its conservation and management. Data of 12 populations were used to assess its genetic diversity and population structure, employing the method of amplified fragment length polymorphism (AFLP). A high level of genetic diversity was detected (H (E) = 0.269) with POPGENE. As revealed by AMOVA analysis, there was moderate variation between pairs of populations with Phi(ST) values ranging from 0.047 to 0.578 and on average 26.97% of the genetic variation occurred among populations. Three main clusters were shown in UPGMA dendrogram using TFPGA, which is consistent with the result of principal coordinate ananlysis (PCO) using NTSYS. Keeping a stable environment is critical for the in situ conservation and management of this rare and endangered plant, and for ex situ conservation it is important to design an integrated germplasm bank.     

Genetic structure of endangered Emmenopterys henryi Oliv. based on ISSR polymorphism and implications for its conservation

Inter-simple sequence repeat (ISSR) markers were used to determine the genetic variation and genetic differentiation of nine populations of Emmenopterys henryi Oliv., an endangered plant endemic to China. Relatively low genetic diversity was detected at population level (the percentage of polymorphic loci P=22.56%, the number of alleles per locus A=1.183+/-0.045, the effective number of alleles per locus A(E)=1.007+/-0.345, Nei's gene diversity h=0.071+/-0.017, Shannon information index I=0.104+/-0.025). However, the genetic diversity at species level was relatively high (P=56.05%; A=1.561+/-0.498, A(E)=1.325+/-0.371, h=0.191+/-0.199, I=0.287+/-0.284). Analysis of molecular variance showed that most of the ISSR variation (68.03%) in E. henryi occurred among populations. The estimated Nm from F (ST )was 0.235. It indicated that the fragmentation and isolation of populations might result from specific evolutionary history and anthropogenic activity. Consequently, genetic drift might play an important role in determining the genetic structure of E. henryi. Conservation strategies for this endangered species are proposed based on the genetic data.     

AFLP和RAPD标记技术在栉孔扇贝遗传多样性研究中的应用比较

AFLP和RAPD标记技术是近年来发展最快的基于PCR基础上的两种DNA标记技术,本文比较了两种标记技术在我国栉孔扇贝群体遗传多样性研究中的应用。共筛选20个RAPD引物和7个AFLP引物组合,检测到AFLP标记的有效等位基因数和平均多态信息量稍低于RAPD标记,但AFLP标记在每单位分析中扩增到的野生和养殖群体的多态性条带数(23．8,24．8)分别高于RAPD标记(5．6,5．6),AFLP多态性检测效率显著高于RAPD标记。AFLP和RAPD两种标记技术所揭示的野生种群与养殖群体间的近交系数、遗传距离两项指标均表明,我国栉孔扇贝养殖群体和野生种群之间尚未出现明显的遗传分化。研究结果表明：RAPD和AFLP这两种标记技术均可用于栉孔扇贝遗传多样性的分析,其分析结果是一致的。     

Genetic diversity of Chinese alligator (Alligator sinensis) revealed by AFLP analysis: an implication on the management of captive conservation

Chinese alligator (Alligator sinensis) is one of the most critically endangered species among 23 extant crocodiles in the world. To prevent the extinction of the species, a captive propagation started at early 1980s, and the total number of alligator was brought up to 10 thousands from dozens of founder in 2000. But several genetic investigations showed those alligators were under an extremely low genetic diversity status with few detectible polymorphic loci. To get more insight into its genetic diversity for the management of captive Chinese alligator, AFLP was adopted to characterize variations in the population. Total of 347 bands were generated from 47 individuals using 3 primer combinations, of which 203 (58.50%) were polymorphic, and 35 AFLP phenotypes were revealed from those individuals. Comparing the results between RAPD and AFLP analysis on almost same sample set clearly indicated that AFLP is more efficient in revealing polymorphic loci, especially in those populations with extremely low genetic diversity. In present three assays, electrophoresis profile also displayed 3 individuals possessing very highly polymorphic AFLP phenotypes that were never been found by RAPD and mtDNA D-loop sequencing, implicating that we should offer these individuals more breeding opportunities to maintain the genetic diversity in the population and restrict those carrying few polymorphic loci from reproduction.     

A Statistical and Comparative Analysis of Genetic Detected by Different Molecular Markers

This paper presented a statistical and comparative analysis of common parameters of plant genetic diversity by using relevant data of 314 wild plant species from 235 published articles. The results indicated that the parameters of genetic diversity revealed by RAPD and AFLP are comparable, but all parameters of genetic variation detected by ISSR, allozyme and SSR are incomparable , which are not comparative with those by RAPD and AFLP. The genetic differentiation value G_st based on Hardy-Weinberg equilibrium is obviously lower than the value Φ_st based on AMOVA analysis, which showed that these two parameters are incomparable as well. Furthermore, the statistical and comparative results of genetic diversity of 179 plant species by RAPD and AFLP indicated that at population level: 1) the genetic diversity of gymnosperm is higher than those of both dicotyledon and monocotyledon of angiosperm, but lower genetic differentiation; 2) the genetic diversity of tree is higher than those of shrub and herb, but lower genetic differentiation; 3) the clonal plant has higher genetic diversity than those reproduce sexnally, and 4) the cross-breeding plant has higher genetic diversity than self- breeding plant; 5 ) the widespread plant species has higher genetic diversity than the rare, endangered or endemic species.     

Genetic Diversity and Population Structure of Yellow Camellia (Camellia nitidissima) in China as Revealed by RAPD and AFLP Markers

Camellia nitidissima, a rare plant but a useful genetic resource for commercial cultivation of ornamental camellias, is distributed in a narrow region of South China and North Vietnam. In this study, RAPD and AFLP markers were used to assess the genetic diversity and population structure of six natural populations of C. nitidissima from Guangxi in South China. Twenty RAPD primers amplified 183 bands, of which 143 bands were polymorphic, and 8 AFLP primer pairs produced 502 bands, of which 364 were polymorphic. Independent as well as combined analyses of the cluster analyses of the RAPD and AFLP fragments showed that the six populations could be classified into two major genetic groups corresponding to the Nanning and Fangcheng areas. The Mantel test revealed significant correlation between the genetic and geographic distances of C. nitidissima populations (r = 0.953, p = 0.036). AMOVA analysis allowed the partitioning of the genetic variation between groups (36.09%), among populations within groups (25.78%), and within populations (38.14%). An understanding of both the genetic diversity and the population structure of C. nitidissima in China can also provide insight into the conservation and management of this endangered species.     

欧洲中部刺槐种源群体等位酶变异研究

从欧洲中部及美国收集了18个刺槐种源种子,以2年生苗木为材料,采用水平切片淀粉凝胶电泳和聚丙烯酰胺凝胶电泳对11个酶系统进行了检测,共发现20个酶位点,其中14个为多态位点,多态性位点百分数为70％。在参加计算的7个酶系统的12个多态性位点中,平均每个位点的等位基因数(A)和平均每位点等位基因有效数(Ae)为2.733和1．794,平均每个位点的实际杂合度(Ho)和预期杂合度(He)为0．368和0．400,固定指数(F)为0．080,绝大多数位点固定指数为正值。各基因位点遗传参数与12个位点平均值进行相关分析表明,Fe-6和Lap-a的大部分群体遗传参数(A、Ae、Ho、He等)与平均值存在紧密的相关关系,其他位点各项遗传参数和总平均值相关不明显,这些位点的重要性可能比相关性较好的位点更大。德国8个种源群体间遗传距离变化在0．09-0．26之间,来自匈牙利的6个种源之间遗传距离很小,绝大部分均在0．11以下;德国的8个种源与匈牙利和斯洛伐克的8个种源之间的遗传距离变化在0．09-0．24之间;来自美国的2个种源,与欧洲的种源间的遗传距离在0．09-0．23之间,这一距离没有超出欧洲种源之间的差异。匈牙利和斯洛伐克的8个种源群体的各项遗传参数均高于德国种源,表明这两个国家的遗传多样性水平较高。同时来自这两个国家的种源群体间变异系数变化在2．92％-9．97％,说明群体之间的遗传差别很小。德国的8个种源群体各项遗传参数相对较低,但群体间变异系数较高,群体间遗传差别较大。以各个国家为群体单位,4个群体间各项遗传参数的变异均较小,变异系数变化在3．861％-5．139％,表明刺槐种源间地理变异模式不明显。     

Microsatellite primers in the Chinese dove tree, Davidia involucrata (Cornaceae), a relic species of the Tertiary

? Premise of the study: The first microsatellite primers were developed for Davidia involucrata, an endangered relic species of the Tertiary in China, to further describe its genetic variability and population structure. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences Containing Repeats (FIASCO) protocol, 15 polymorphic microsatellite loci were isolated and characterized in 20 individuals from the germplasm collections of D. involucrata at the Hunan Forest Botanical Garden. High levels of polymorphism were revealed, with the total number of alleles per locus and the number of alleles per locus per individual ranging from two to 13 and from one to six, respectively. ? Conclusions: The multibanded patterns of microsatellite loci obtained in the current study confirmed that D. involucrata might be a polyploid species. The primers will be useful for studies of genetic diversity and for guiding conservation strategies for D. involucrata.     

AFLP and ISSR analysis reveals high genetic variation and inter-population differentiation in fragmented populations of the endangered Litsea szemaois (Lauraceae) from Southwest China

Litsea szemaois (Lauraceae) is an endemic and endangered species from the tropical rain forests of Xishuangbanna, southern Yunnan, SW China, but habitat fragmentation, especially exacerbated by rubber planting, has caused a decline in population size of the species. AFLP and ISSR were used to investigate the genetic diversity and population structure of eight populations from across its known distribution. Three AFLP and ten ISSR primer combinations produced a total of 203 and 77 unambiguous and repeatable bands respectively, of which 164 (80.8%) and 67 (87.0%) were polymorphic for the two markers. These two markers showed that Litsea szemaois exhibits comparatively high genetic diversity at species level (heterozygosity (hs) = 0.2109) relative to some other Lauraceae. Most of the genetic variation was partitioned within populations, but genetic differentiation between populations was significant and relatively high (Φ _st = 0.2420, θ^B = 0.1986) compared with other tropical plants. The genetic characteristics of L. szemaois may be related to its outbreeding system, insect pollination and fragmented distribution. Because L. szemaois is dioecious and slow to mature, ex situ conservation across its genetic diversity is unlikely to succeed, although seedlings grow well under cultivation. Thus, in situ conservation is very important for this endangered species, especially as only 133 adult individuals are known in the wild. In particular, the Nabanhe and Mandian populations should be given a high conservation priority due to their higher genetic diversity, larger population size and better field condition, but wider sampling is required across all populations to determine additional areas with significant genetic conservation value.     

Microsatellite primers in the endangered quillwort Isoetes hypsophila (Isoetaceae) and cross-amplification in I. sinensis

? Premise of the study: The first microsatellite primers were developed for Isoetes hypsophila, an endangered quillwort species endemic to the Qinghai-Tibetan Plateau in China, to further describe its genetic variability and population structure. We also examined their cross-amplification in a congeneric species, I. sinensis. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol, nine microsatellite loci were isolated and characterized in 32 samples from four natural populations of I. hypsophila. The primers amplified di- and hexanucleotide repeats with three to 11 alleles per locus. Seven of nine primers were cross-amplified in I. sinensis with two to seven alleles per locus. ? Conclusion: The microsatellite loci primers will be useful for studies of genetic diversity and gene flow in natural populations of Isoetes species.